PLCconverts phosphatidylinositol-(4,5)-bisphosphate to the second messengers inositol-(1,4,5)-triphosphate and diacylglycerol, promoting Ca2+ flux and PKC activation, respectively. transphosphorylation upon BCR aggregation (13). It is unclear whether the signals transmitted by unligated receptors are qualitatively distinct from those of ligated receptors or merely represent a quantitative difference. An important step in BCR signaling is the phosphorylation of the coreceptor CD19 (14). CD19 physically associates with the BCR through intracellular and extracellular motifs (15C17). (CD19 has also been shown to facilitate pre-BCR signaling (18, 19)). BCR ligation leads to the recruitment by CD19 of PI3K via its p85regulatory subunit, the generation of lipid products such as phosphatidylinositol-(3,4,5)-trisphosphate (PIP3), and the attendant recruitment to the plasma membrane of pleckstrin homology (PH) domain-containing proteins, such as phospholipase C (PLC)protein (25), the EBV LMP2A protein (26, 27), or an designed sIg molecule devoid of Ag specificity (22). During bone marrow development, transgene-enforced expression of prerearranged IgH or IgH/L combinations can suppress endogenous rearrangements, demonstrating a feedback regulation process; however, autoreactive receptors that presumably generate a distinct BCR-mediated signal fail to suppress recombination and promote instead ongoing rearrangement that often leads to receptor editing (reviewed in Ref. 28). These results suggest that in immature B cells an unligated BCR promotes a signal that regulates V(D)J recombination, whereas Cdh5 a cross-linked receptor promotes a distinct signal. Furthermore, studies in which the sIg is usually inducibly lost from immature B cells suggest that this suppression of recombination, along with the loss of expression of maturation markers, is usually reversible for some time (24). An important aspect of the regulation of L chain recombination involves the transcription rate of and clone that appears to carry transcriptional control regions for both RAG1 and RAG2 (32). Mice were maintained in The Scripps Research Institute Animal Resources facility; all of these studies have been reviewed and approved by the relevant The Scripps Research Institute institutional animal care and use review committee. Cell culture and stimulation Immature Cadherin Peptide, avian B cells were either isolated directly from the bone marrow of 3-83 mice, or expanded in IL-7 cultures, and stimulated with BCR and control mAbs at 10 centrifugation for 10 min at 4C, and supernatants were stored at C70C. After reducing PAGE, transfer to nylon membranes was conducted using the X Cell II Blot Module (Invitrogen Life Technologies). Primary Abs used were: p50/p105 (sc-114), p65 (sc-372), c-(sc-71), I(sc-371), p-I(sc-8404), Akt1 Cadherin Peptide, avian (sc-1618), Btk (sc-1696), and cyclin D2 (sc-593) from Santa Cruz Biotechnology; phospho-Btk (Tyr223), phospho-Akt (Thr308), phospho-PLC(37) and TAT-in the may be an under-glycosylated CD19 biosynthetic intermediate). To extend this analysis in vivo, we assessed CD19 levels in freshly isolated bone marrow B cells that were either innocuous (3-83 Tg on a nondeleting H-2d background) or autoreactive (3-83 Tg central-deleting H-2k background). In B cells around the autoreactive background, levels of both surface CD19 and intracellular CD19 were down-modulated significantly (Fig. 2). We conclude that CD19 tyrosine phosphorylation at Y513 correlates with B cell-positive selection, whereas in the context of unfavorable selection, developing B cells carry reduced levels of CD19 Cadherin Peptide, avian that are hypophosphorylated. Open in a separate window Physique 1 Innocuous BCR signal promotes protein tyrosine phosphorylations that are inhibited by prolonged BCR cross-linking. IL-7-cultured 3-83 Tg bone marrow B cells were treated after IL-7 withdrawal for the indicated occasions with either anti-BCR or control Abs. (inhibitor, IC87114 (45); it too promoted RAG expression in a dose-dependent manner (Fig. 3catalytic component (IC87114) on RAG1 and RAG2 expression. Effect of p85 deficiency on RAG expression in vivo To further probe the role of PI3K in B cell positive-selection, 3-83 BCR Tg mice were bred to p85deficiency as an explanation for these results. Consistent with the elevated RAG expression of the p85function appears to be important for the innocuous BCR-mediated suppression of RAG expression in vivo. Open in a separate window Physique 4 Analysis of RAG expression and L chain gene recombinations in p85-deficient 3-83 bone marrow B cell cultures and primary ex vivo cells. transcription factors regulate RAG expression, we tested the effects of p85deficiency around the spontaneous and sustained BCR-induced activation of NF-deficiency was indeed correlated with elevated NF-and activity was high even in the absence of BCR ligation. Elevated NF-was generally correlated with reduced levels of cytoplasmic Iand increases of p65 levels in the nucleus (Fig. 5components found in the nucleus had a distinctly slower electrophoretic mobility (Fig. 5and (TAT-Isuperrepressor protein (29). Consistent with the prediction that.